Vol.:(0123456789) 1 3 Journal of Proteins and Proteomics https://doi.org/10.1007/s42485-019-00007-9 ORIGINAL ARTICLE Purifcation of dual‑functioning chitinases with hydrolytic and antifreeze activities from Hippophae rhamnoides seedlings Bhavana Sharma 1  · Ravi Gupta 2  · Dinabandhu Sahoo 3  · Renu Deswal 1 Received: 4 January 2019 / Revised: 5 February 2019 / Accepted: 7 February 2019 © Springer Nature Singapore Pte Ltd. 2019 Abstract Chitinases are glycosyl hydrolases which hydrolyse β-1,4-glycosidic bonds between N-acetylglucosamine residues of chitin. Seabuckthorn (Hippophae rhamnoides), a cold desert plant, is a storehouse of many cold-tolerant proteins including dual- functioning antifreeze proteins (AFPs) possessing both hydrolytic and antifreeze activities. Herein, we report the purifcation and characterization of antifreeze chitinases from seedlings grown in laboratory conditions. Chitin-afnity chromatography led to homogenous purifcation of two acidic chitinases HrS CHT1a (33 kDa) and HrS CHT1b (38 kDa) from seedlings. Antifreeze activity of purifed AFPs was confrmed by the formation of hexagon-shaped ice crystals using nanolitre osmom- eter. Similarly, sucrose sandwich splat assay also confrmed their ice recrystallization inhibition activity (1.6-fold decrease in mean ice crystals). The chitinase activity of AFPs was confrmed by chitin hydrolytic assay where higher activity (1.8- fold) was observed in HrS CHT1b (500 U/mg) than HrS CHT1a (222 U/mg). MS identifcation showed homology of HrS CHT1b with provicilin while HrS CHT1a was identifed as uncharacterized protein. In silico analysis showed that purifed AFPs difer signifcantly in biochemical properties which suggests their diferent physiological roles. Protein association network analysis using string showed interaction of HrS CHT1b with enzymes involved majorly in pathogenic protection (pectinesterase, glycosyl hydrolase protein with chitinase domain). However, HrSCHT1a showed interaction with proteins associated with growth and energy regulation (glycine and purine synthesis, vitamin B metabolism) thereby indicating dif- ferential functional roles of both the chitinases. Conserved domain analysis also supported that these AFPs are multifunctional and exhibit diferential regulatory roles in enabling the plant growth and defense responses. Further validation of these targets may open gates for commercial utilization of this plant growing abundantly in the Himalayan regions of India for protection of freeze-susceptible crops or biomedical applications. Keywords Antifreeze activity · Chitin-afnity chromatography · Dual functioning AFPs · Hippophae rhamnoides · Hydrolytic assay Introduction Plants are constantly exposed to diverse abiotic stresses including food, drought, temperature fuctuations, salinity, mineral defciency and toxicity which restrict their growth, productivity, cultivation, survival and geographical distribu- tion. Abiotic stress conditions make the plants more prone to pathogenic infections owing to increased emergence of virulent and broad host range pathogens. Low-temperature stress is one of the major environmental factors afecting the agricultural crop yield worldwide. Plants have complex cold/freezing stress tolerance mechanisms including antifreeze proteins (AFPs) to pro- tect themselves from cold/freezing stress. Cold-responsive dual-functioning AFPs comprise a diverse family of proteins * Renu Deswal rdeswal@botany.du.ac.in; deswalr@hotmail.com 1 Molecular Physiology and Proteomics Laboratory, Department of Botany, University of Delhi, Delhi 110007, India 2 Department of Plant Bioscience, College of Natural Resources and Life Sciences, Pusan National University, Miryang, South Korea 3 Department of Biotechnology, Institute of Bioresources and Sustainable Development, Imphal, Manipur, India